Our overall goal is to determine how circadian rhythms of behavior are generated. In previous work on this project we identified and characterized molecular mechanisms of the endogenous circadian clock in Drosophila. We also initiated studies to identify the mechanisms that carry time of day cues from the clock and transmit them through the brain. Specifically, we identified output neurons that are required for rhythmic rest:activity and connect anatomically to central clock neurons. These output neurons include the Dorsal Neuron 1 (DN1) group, which contains a clock, and two non-clock clusters in the pars intercerebralis (PI), a region of neurosecretory cells equivalent to the mammalian hypothalamus. The two PI clusters that regulate circadian rhythms of rest:activity secretes the neuropeptides DH44 and SIFamide respectively. We found that DH44 is required for behavioral rhythms, but a role for SIFamide has not been determined yet. Interestingly, the DN1s have also been associated with the function or expression of known circadian output molecules, narrow abdomen and unpaired. In addition, our preliminary data indicate that another output molecule, Neurofibromatosis 1 (NF1), is required in DN1s for rest:activity rhythms. We also find that neural activity cycles with a 24 hour rhythm in DN1 and DH44 cells. Thus, we have started to place molecular components in the cellular substrates we identified, and develop assays to monitor the transmission of rhythmic signals through the network. We hypothesize that time-of-day cues generated in central clock cells are transmitted through DN1s to drive rhythmic activity in the PI, which then controls rest:activity rhythms through release of specific neuropeptides. We propose to: (1) Address the significance of rhythmic activity in the DN1s and determine which clock cells and output molecules are required for this rhythm. (2) Identify the upstream components in clock cells that drive rhythmic activity in the DH44 cells, and determine if DH44 acts rhythmically. (3) Address a role for SIFamide in rest:activity rhythms and identify other PI molecules relevant for rest:activity rhythms. Together these studies are expected to provide a comprehensive understanding of the molecular network and cellular circuit that generates a behavioral rhythm. Given known conservation of clock mechanisms and molecules from flies to humans, these studies will likely be relevant for our understanding of human rhythms, which are critical for normal behavior and physiology. These studies will also provide general insight into the maintenance and function of neural circuits, which are impaired in several neurological disorders.